title: Direct measurements of oscillatory glycolysis in pancreatic islet β-cells using novel fluorescence resonance energy transfer (FRET) biosensors for pyruvate kinase M2 activity.
abstract: Pulses of insulin released from pancreatic β-cells maintain blood glucose in a narrow range, although the source of these pulses is unclear. We and others have proposed that positive feedback mediated by the glycolytic enzyme phosphofructokinase-1 (PFK1) enables β-cells to generate metabolic oscillations via autocatalytic activation by its product fructose 1,6-bisphosphate (FBP). Although much indirect evidence has accumulated in favor of this hypothesis, a direct measurement of oscillating glycolytic intermediates has been lacking. To probe glycolysis directly, we engineered a family of inter- and intramolecular FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PKAR; pyruvate kinase activity reporter), which multimerizes and is activated upon binding FBP. When introduced into Min6 β-cells, PKAR FRET efficiency increased rapidly in response to glucose. Importantly, however, metabolites entering downstream of PFK1 (glyceraldehyde, pyruvate, and ketoisocaproate) failed to activate PKAR, consistent with sensor activation by FBP; the dependence of PKAR on FBP was further confirmed using purified sensor in vitro. Using a novel imaging modality for monitoring mitochondrial flavin fluorescence in mouse islets, we show that slow oscillations in mitochondrial redox potential stimulated by 10 mm glucose are in phase with glycolytic efflux through PKM2, measured simultaneously from neighboring islet β-cells expressing PKAR. These results indicate that PKM2 activity in β-cells is oscillatory and are consistent with pulsatile PFK1 being the mediator of slow glycolytic oscillations.
authors.affiliation: From the Department of Pharmacology and Brehm Center for Diabetes Research, University of Michigan Medical School, Ann Arbor, Michigan 48105.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Merrins
authors.forename: Aaron R
authors.initials: AR
authors.lastname: Van Dyke
authors.forename: Anna K
authors.initials: AK
authors.lastname: Mapp
authors.forename: Megan A.
authors.initials: MA
authors.lastname: Rizzo
authors.forename: Leslie S
authors.initials: LS
authors.lastname: Satin
mesh_terms: D000818:Animals; D001683:Biological Clocks / Q000502:physiology*; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005635:Fructosediphosphates / Q000235:genetics / Q000378:metabolism; D006019:Glycolysis / Q000502:physiology*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000201:enzymology*; D008297:Male; D051379:Mice; D010084:Oxidation-Reduction; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000235:genetics / Q000378:metabolism*
chemical_list: D005635:Fructosediphosphates; D010732:Phosphofructokinase-1; D011770:Pyruvate Kinase; C029063:fructose-1,6-diphosphate
keywords: Beta Cell; Flavin; Fluorescence Resonance Energy Transfer (FRET); Glycolysis; Mitochondrial Metabolism; NADH; Pancreatic Islets